Method and kit for determining the genome integrity and/or the quality of a library of DNA sequences obtained by deterministic restriction site whole genome amplification

Abstract

A method for determining the integrity of the genome of a sample and/or the quality of a library of DNA sequences obtained by deterministic restriction site whole genome amplification can include (a) amplifying the library of DNA sequences to produce first, second, and third PCR products each of a different size from 50 bp to 1000 bp, by PCR using at least one first primer pair, one second primer pair and one third primer pair, the primer pairs each hybridizing to a DNA sequence of the library having a length from 1000 bp to 5000 bp and corresponding to a sequence of the genome located respectively on a first, second and third chromosome arm; (b) detecting the first, second and third PCR products; (c) correlating the presence of the first, second and third PCR products with the integrity of the genome of the sample and/or the quality of the library.

The invention claimed is: 
     
       1. A method for determining the integrity of the genome of a sample to be analysed by whole genome amplification (WGA) and/or the quality of a library of DNA sequences obtained by deterministic restriction site whole genome amplification (DRS-WGA) of the genome of the sample comprising the steps of:
 (a) providing a sample having a genome 
 (b) obtaining a library of DNA sequences by deterministic restriction site whole genome amplification (DRS-WGA) of the genome of the sample; 
 (c) amplifying the library of DNA sequences by PCR using:
 at least one first primer pair which hybridizes to a DNA sequence of the library having a length from 1000 bp to 5000 bp and encompassing the D5S2117 region of chromosome 5q, 
 at least one second primer pair which hybridizes to a DNA sequence of the library having a length from 1000 bp to 5000 bp and encompassing exons 2 and 3 of the TRP53 gene of the genome, and 
 at least one third primer pair which hybridizes to a DNA sequence of the library having a length from 1000 bp to 5000 bp and encompassing the KRT19 pseudo-gene 1 of the genome, 
 
 wherein the step of amplifying giving rise to a first PCR product from 50 bp to 1000 bp, a second PCR product from 50 bp to 1000 bp having a size other than the first PCR product, and a third PCR product from 50 bp to 1000 bp having a size other than the first and second PCR products; and 
 (d) detecting the first, second and third PCR products, 
 wherein the presence of one or more of the first, second and third PCR products corresponds to a level of the integrity of the genome of the sample and/or the quality of the library of DNA sequences. 
 
     
     
       2. The method according to  claim 1 , wherein a forward primer of the at least one first primer pair is SEQ ID NO:4 and a reverse primer of the at least one first primer pair is SEQ ID NO:3. 
     
     
       3. The method according to  claim 2 , wherein step (c) further uses at least one fourth primer pair which hybridizes to a DNA sequence of the library having a length from 80 bp to 300 bp, the step of amplifying giving rise to a fourth PCR product from 50 bp to 200 bp having a size other than the first, the second and the third PCR products;
 step (d) further comprises detecting the fourth PCR product; 
 wherein the presence of one of more of the first, second, third, and fourth PCR product corresponds to the level of the integrity of the genome of the sample and/or the quality of the library of DNA sequences. 
 
     
     
       4. The method according to  claim 3 , wherein the DNA sequence of the library to which the at least one fourth primer pair hybridises encompasses Codon 12/13 of the KRAS gene. 
     
     
       5. The method according to  claim 4 , wherein a forward primer of the at least one fourth primer pair is SEQ ID NO:17 and a reverse primer of the at least one first primer pair is SEQ ID NO:18. 
     
     
       6. The method according to  claim 1 , wherein a forward primer of the at least one second primer pair is SEQ ID NO:6 and a reverse primer of the at least one second primer pair is SEQ ID NO:5. 
     
     
       7. The method according to  claim 1 , wherein a forward primer of the at least one third primer pair is SEQ ID NO:8 and a reverse primer of the at least one third primer pair is SEQ ID NO:7. 
     
     
       8. The method according to  claim 1 , wherein the sample consists of 5 cells or less. 
     
     
       9. The method according to  claim 1 , wherein the sample is a single cell. 
     
     
       10. The method according to  claim 1 , wherein determining the integrity of the genome of the sample allows to determine the biological status of the cell/cells of the sample.